PROJECT 1: SYSTEMATIC IDENTIFICATION OF HOST NETWORKS IN INFECTIOUS DISEASE PATHOGENESIS SUMMARY The Centers for Disease Control and Prevention (CDC) estimates that more than 2 million people acquire a serious drug-resistant bacterial infections each year, with at least 23,000 deaths resulting. These numbers will increase dramatically as the frequency of multidrug resistant bacteria rise and infections spread worldwide. Unfortunately, the production of novel classes of antibiotics has stagnated since the 1960s, thus underscoring a critical need for the development of alternate approaches that can be used to treat infection. There has been a new push for the development of host-directed therapies for treatment of infectious diseases as they are expected to be less susceptible to drug-resistance. In addition, recent work has revealed that while similar proteins may not be targeted by different pathogens, the same functional pathways are often hijacked and re-wired during the course of infection. Thus, drugs that target host pathways, rather than individual pathogen factors, may represent improved targets for treatment. For these reasons, the study of infectious disease is becoming increasingly dependent on knowledge of host biological networks of multiple types, including physical interactions among proteins, which allow for deconstruction of functional pathways. Here we propose to systematically identify the protein networks that drive pathogenesis in clinically relevant model systems. Coupled with functional validation and high-resolution structural analysis of key pathogen-host interactions and complexes, we anticipate major insights into the underlying biology of pathogenesis, as well as the potential to unravel novel vulnerabilities of therapeutic relevance. To this end, we are targeting hundreds of pathogen encoded genes from Mycobacterium tuberculosis, Staphylococcus aureus, and Chlamydia trachomatis, and subjecting them to affinity purification mass spectrometry (AP-MS) in a panel of clinically relevant immune cell lines (Aim 1). To complement these data we will perform proteome wide quantitative profiling of phosphorylation, ubiquitylation and protein abundance levels over a time course of pathogen infection (Aim 2). In Aim 3, we will use a suite of structural characterization methods, including X-ray crystallography, cryogenic electron microscopy (cryo-EM) and cross-linking mass spectrometry (XL-MS) to structurally characterize therapeutically actionable protein complexes and signaling nodes. These aims will inform the selection of host target proteins for in vivo validation in Aim 4, in which we will generate knockout mice and subject them to infection to test for increased resistance to bacterial pathogens. Successful completion of this this work will not only significantly enrich our limited understanding of host-pathogen networks interactions, but it will also identify novel therapeutic opportunities for these three pathogens. Additionally, the development of this platform will yield an integrated systems-to-structure pipeline that can be extended to many pathogen types, and will aid in the rational selection of therapeutic targets with greater precision and speed. 1